RAPID ASSAY PROCESSING BY INTEGRATION OF DUAL-COLOR FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY - HIGH-THROUGHPUT SCREENING FOR ENZYME-ACTIVITY

Dual-color fluorescence cross-correlation spectroscopy (dual-color FCS ) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies, In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary b iotechnology is described, This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depen d on the characterization of diffusion parameters that is the prerequi site for conventional fluorescence correlation spectroscopy. Dual-colo r FCS parameters were optimized to achieve the shortest analysis times , A simulated HTS with homogeneous assays for different restriction en donucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or- no decisions within analysis times of about 1 s per sample, RAPID FCS combines these short analysis times with the development of fast and f lexible assays resulting in sensitive, homogeneous fluorescence based assays, where a chemical linkage between different fluorophores is eit her cleaved or formed, or where differently labeled molecules interact by noncovalent binding, In principle, assay volumes can be reduced to submicroliters without decreasing the sign al strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology, RAP ID FCS can accurately probe 10(4) to 10(5) samples per day, and possib ly more, In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where ra re and specific binding or catalytic properties have to be screened in large numbers of samples.